Isolated and expanded cartilage-forming cells have been used for implantation since several decades. The in vitro expansion to obtain a sufficient amount of cells however may lead to cells which upon implantation result in cells death, scar tissue or hyperdifferentiated cells. This is especially the case for cartilage repair where chondrocytes culture, upon implantation often produces fibrous or bone-like material instead of the envisaged hyaline cartilage.
Attempts have been made to cultivate chondrocyte cells in a three dimensional matrix wherein the extracellular matrix of cartilage is mimicked. A variety of gels has been used wherein cell are cultivated. For example, the prior art discloses beads with a core of alginate and a coating of chitosan (Babister et al. (2008) Biomaterials 29, 58-65).
Methods to prepare hydrogel beads can be divided in three groups. A first type relates to porous matrices which are populated by cells. Herein the matrices have a large pore size to allow the migration of cells into the matrix. Li et al. (2008) J. Biomed. Mater. Res. A. 86, 552-559, describe a porous scaffold containing 2.4% alginate/2.4% chitosan which is subsequently seeded with cells.
As the cells migrate from the outside into the interior of the matrix, these matrices often have many cells at the outer cells and a low amount of cells at the interior.
In a second type of methods cells are mixed with a constituent of the matrix whereafter the matrix is being formed. Bernstein et al. (2009) Biotechnol. Prog. 25, 1146-1152, describe methods wherein beads with 2.5% alginate and 1.4% chitosan are formed. The cartilage therein is described as being of low quality.
In a third method, as illustrated in WO2007/135114, cells are encapsulated in a gelling solution obtained from an aqueous solution of a mixture of a polysaccharide such as alginate and an oligosaccharide derivative of chitosan highly branched. The aqueous solution is gelled with gelling agents with the aim to encapsulate the cells.
In each type of methods the choice of hydrogel has a strong impact on the phenotype of the cells, and subsequently on the quality of the implant. Particularly in the hydrogel of WO2007/135114, oligosaccharide derivative of chitosan has a degree of derivatization of at least 40% and the polysaccharide mixture or alginate mixture has an ionic strength which is not optimal for keeping cells alive. Further this 3D matrix fails to keep chondrocyte phenotype stable as shown by an increase of cell proliferation and a decrease of aggrecan synthesis.
There remains a need for further improvements in the selection of matrix material and in the methods to produce cellularised implants.